The present invention relates generally to cell monitoring and ligand detection and evaluation, and more particularly to a method of monitoring the level of a primary ligand in a cell or a method of evaluating a molecule for primary ligand-binding activity, using a green fluorescent protein fusion complex that exhibits ligand-dependent fluorescence resonance energy transfer.
Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
The activity of a cell is influenced and regulated by many molecules. Many cells have receptors which activate pathways within the cell, such as the cAMP pathway or the phosphoinositide pathway. Such receptors are often activated by a ligand. When the ligand binds to its receptor, the receptor activates the pathway. Other molecules influential in a cell include ions such as calcium ions. In an attempt to understand the molecular biology of a cell, the ways in which cells interact, or even causes of cell death, more knowledge is continually needed regarding the distribution and/or pattern of expression of various molecules within a cell or within a tissue or organ.
One example of such a molecule is calmodulin. Calmodulin modulates the activities of a large and constantly increasing number of known target proteins (Bredt and Snyder 1990; Cho et al. 1992; Edelman et al. 1996; Crivici and Ikura 1995; Kink et al. 1990; Lu and Means 1993; Manalan and Klee 1984; Means et al. 1991; Wang et al. 1996). Through their actions it participates in the regulation of most processes in the cell including motility, axonal transport, synaptic transmission, ion homeostasis, cell cycle progression, gene expression and apoptosis. It is of interest to determine whether maximal and resting free (Ca2+)4-calmodulin levels vary among cell types, or at different points in the cell cycle, which has been shown to be associated with cyclic changes in total calmodulin levels. It would also be of interest to investigate spatio-temporal changes in calmodulin activity during cellular events that involve it.
It would also be of interest to determine maximal and resting levels of other molecules and ligands, in various cell types, or at different points in the cell cycle, and well as to determine spatio-temporal changes in ligand-binding activity during cellular events that involve it.
To this end, the subject invention provides a method of monitoring the amount of a primary or secondary ligand in a cell. The method first involves the construction of a green fluorescent protein (GFP) complex. The green fluorescent protein complex comprises a first green fluorescent protein which is excited at a first wavelength and which emits fluorescence at a second wavelength; a primary ligand-binding peptide having an amino terminal end and a carboxy terminal end, the amino terminal end of the primary ligand-binding peptide being covalently attached to the first green fluorescent protein; and a second green fluorescent protein which is excited at the second wavelength and which emits fluorescence at a third wavelength, the carboxy terminal end of the primary ligand-binding peptide being covalently attached to the second green fluorescent protein. The constructed green fluorescent protein complex is then introduced into a cell, and a base amount of fluorescence emission at the third wavelength when the cell is excited at the first wavelength is determined. This determination provides a control or base amount of fluorescence resonance energy transfer between the two green fluorescent proteins of the green fluorescent protein complex. The amount of fluorescence emission over time at the third wavelength when the cell is excited over time at the first wavelength is determined. Next, a comparison is made of the amount of fluorescence emission over time to the base amount of fluorescence emission, wherein an amount of fluorescence emission less than the base amount of fluorescence emission indicates an increase in the amount of the primary ligand in the cell. In particular, the primary ligand present in the cell has bound to the primary ligand-binding peptide, changing the distance between the two GFP molecules and altering fluorescence resonance energy transfer (FRET) between them. Changes in the amount of fluorescence emission over time can be used to monitor changes in the amount of the primary ligand in the cell over time.
By quantitating the amount of fluorescence emission over time using the subject method, one can estimate the amount of the primary ligand in the cell over time.
The subject invention further provides a method for monitoring the amount of a secondary ligand in the cell, wherein the secondary ligand binds to the primary ligand and is necessary for binding of the primary ligand to the primary ligand-binding peptide. This method is as recited above, except the method further comprises introducing a known concentration of the primary ligand into the cell prior to determining the base amount of fluorescence emission. When the amount of fluorescence emission is less than the base amount of fluorescence emission, it is an indication of the presence of the secondary ligand. As above, changes in the amount of fluorescence emission over time indicate changes in the amount of the secondary ligand in the cell over time.
The invention further provides a method of screening a peptide for primary ligand-binding activity. The method comprises constructing a green fluorescent protein complex. In this embodiment, the green fluorescent protein complex comprises the first green fluorescent protein and the second green fluorescent protein as described above, but they are covalently attached by a peptide having an amino terminal end and a carboxy terminal end, the amino terminal end of the peptide being covalently attached to the first green fluorescent protein and the carboxy terminal end of the peptide being covalently attached to the second green fluorescent protein. The green fluorescent protein complex is introduced into a cell in the absence of the primary ligand, and a determination of the base amount of fluorescence emission at the third wavelength is made when the cell is excited at the first wavelength. The candidate primary ligand is then added to the cell, and a determination of the amount of fluorescence emission at the third wavelength is made when the cell is excited at the first wavelength. A comparison is then made of the amount of fluorescence emission to the base amount of fluorescence emission, wherein an amount of fluorescence emission less than the base amount of fluorescence emission indicates that the molecule has primary ligand-binding activity. Alternatively, this method of screening a peptide for primary ligand-binding activity can be performed in vitro in, for example, a test tube or well. The green fluorescent protein complex is expressed in a cell and then purified from the cell by conventional protein purification techniques (such as affinity chromatography). The purified protein complex is placed in a test tube or well and a determination is made of the base amount of fluorescence emission at the third wavelength when the purified protein complex is excited at the first wavelength. The candidate primary ligand is then added to the test tube or well, and a determination is made of the amount of fluorescence emission at the third wavelength when the purified protein complex (in the presence of purified primary ligand) is excited at the first wavelength. As in the previous embodiment, a comparison is then made of the amount of fluorescence emission to the base amount of fluorescence emission, wherein an amount of fluorescence emission less than the base amount of fluorescence emission indicates that the peptide has primary ligand-binding activity.
Further provided is another method of screening a molecule for the ability to bind a primary ligand in competition with the primary ligand-binding peptide where the constructed green fluorescent protein complex comprises the first green fluorescent protein and the second green fluorescent protein covalently attached by a primary ligand-binding peptide having an amino terminal end and a carboxy terminal end. The amino terminal end of the primary ligand-binding peptide is covalently attached to the first green fluorescent protein and the carboxy terminal end of the primary ligand-binding peptide is covalently attached to the second green fluorescent protein. The green fluorescent protein complex is introduced into a cell or test vesicle (such as a test tube or test well) with the primary ligand, and a determination is made of the base amount of fluorescence emission at the third wavelength when the cell or contents of the test vesicle are excited at the first wavelength A molecule, such as a peptide, is then added to the cell or test vesicle, and a determination is made of the amount of fluorescence emission at the third wavelength when the cell or contents of the test vesicle is excited at the first wavelength. A comparison is then made of the amount of fluorescence emission to the base amount of fluorescence emission, wherein an amount of fluorescence emission greater than the base amount of fluorescence emission indicates that the molecule has primary ligand-binding activity. In particular, the molecule has bound the primary ligand thereby preventing the primary ligand from interfering with energy transfer between the two GFPs (the primary ligand does this by binding to the primary ligand-binding peptide that covalently attaches the two GFPs).
The invention further provides a green fluorescent protein complex which comprises a first green fluorescent protein which is excited at a first wavelength and which emits fluorescence at a second wavelength; a primary ligand-binding peptide which reversibly binds a primary ligand, the primary ligand-binding peptide having an amino terminal end and a carboxy terminal end, the amino terminal end of the primary ligand-binding peptide being covalently attached to the first green fluorescent protein; and a second green fluorescent protein which is excited at the second wavelength and which emits fluorescence at a third wavelength, the carboxy terminal end of the primary ligand-binding peptide being covalently attached to the second green fluorescent protein. When the green fluorescent protein complex is excited at the first wavelength, the amount of fluorescence emission at the third wavelength is reduced when the primary ligand-binding peptide is bound to primary ligand as compared to the amount of fluorescence emission at the third wavelength when the primary ligand-binding peptide is not bound to the primary ligand. Also provided is a cell which expresses the green fluorescent protein complex.